T cells were purified from peripheral blood mononuclear cells (PBMC) of healthy donors or of patients with gastric carcinoma. PBMC were isolated by density gradient centrifugation (Lymphosep, Aurogene) and then were activated by anti-Biotin MACSi Bead Particles loaded with anti-CD2, anti-CD3, and anti-CD28 antibodies (Miltenyi Biotec) for 24 hrs. PBMC were maintained in culture medium + human recombinant IL-2 (100 U/mL, Miltenyi Biotech) for a further 24 hrs and then transduced with MET-CAR-LVs (MOI 10) for 8 hrs in the presence of polybrene (8 μg/mL). Not-transduced T cells (NTD) were used as a paired control. CAR-T or NTD-T cells were expanded for a maximum of 3 weeks in the presence of IL-2 (100 U/mL). Transduction efficiency (evaluated by eGFP expression) ranged from 30 to 70% of the total population.
Anti biotin macsibead particles
Manufactured by Miltenyi Biotec
Sourced in United States, Germany
Anti-biotin MACSiBead particles are laboratory-scale magnetic particles designed for isolating and enriching biotin-labeled cells or molecules. The particles contain immobilized anti-biotin antibodies that bind to biotin-labeled targets. This allows for efficient separation and purification of the labeled samples using a magnetic field.
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Lab products found in correlation
Vpd450 violet proliferation dye, by BD (3 mentions)
Cd4 t cell isolation kit, by Miltenyi Biotec (3 mentions)
Carboxyfluorescein succinimidyl ester (cfse), by Miltenyi Biotec (2 mentions)
Celltrace violet, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Glutamax, by Thermo Fisher Scientific (2 mentions)
Recombinant human il 2, by Miltenyi Biotec (1 mentions)
Anti biotin macsibead, by Miltenyi Biotec (1 mentions)
T cell activation expansion kit, by Miltenyi Biotec (1 mentions)
Golgi plug transport inhibitor, by BD (1 mentions)
Monensin, by Thermo Fisher Scientific (1 mentions)
Cytofix cytoperm fixation permeabilization kit, by BD (1 mentions)
Navios ex, by Beckman Coulter (1 mentions)
Anti cd28 clone 15e8, by Miltenyi Biotec (1 mentions)
Anti cd3 clone okt3, by Miltenyi Biotec (1 mentions)
Macs t cell isolation kits, by Miltenyi Biotec (1 mentions)
Macs mdsc isolation kit, by Miltenyi Biotec (1 mentions)
Interleukin 2 (il 2), by BioLegend (1 mentions)
Carboxyfluorescein succinimidyl ester (cfse), by Tree Star (1 mentions)
Carboxyfluorescein succinimidyl ester (cfse), by Thermo Fisher Scientific (1 mentions)
19 protocols using anti biotin macsibead particles
1
Generation of MET-CAR Modified T Cells
2
T cell Deactivation by MSC-derived EVs
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Proliferation of activated T cells was assessed as a measure of T cell deactivation. Initially, for positive controls 5 × 104 MSCs per well were cultured in 96-well plates for 24 h at 37 °C. CD4+ T cells were purified from spleens and lymph nodes (popliteal/inguinal) of healthy C57Bl/6 mice using the CD4+ T Cell Isolation Kit (Miltenyi Biotec Ltd., Bisley, UK) following manufacturer’s instructions. T cells were seeded at a density of 5.0 × 105/well in 250 μL RPMI medium with 10% FBS and cultured for 5 days with MSCs (ratio 10:1) or with EV-NormO2, EV-2%O2 or EV-Pro-Inflam (ratio equivalent to secretions from 10:1 cells) (n = 8). T cells alone served as control. Cells were activated using anti-Biotin MACSiBead Particles (Miltenyi Biotec Ltd., Bisley, UK) (ratio 2:1). EV treatments were refreshed after 2 days of culture. Polarisation was assessed as above and proliferation measured through reduction in signal intensity using VPD450 Violet proliferation dye (BD Biosciences) [3 (link)]. Data were analysed using a 2-way repeated-measures ANOVA with Bonferroni multiple comparison test post hoc.
3
Antibody Conjugation to Magnetic Beads
4
Activating T Cells for Skin Reconstruction
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Mononuclear cells were isolated from peripheral blood obtained from six healthy donors by Ficoll-Hypaque density gradient centrifugation, followed by three periods of adhesion (1 h, 2 h and overnight) on a plastic support to isolate the adherent monocytes / macrophages from the remaining T cells in suspension. All patients, different from those whose skin cells were extracted, were given adequate information to provide written consent. T cells were then activated with anti-biotin MACSiBead Particles loaded with biotinylated antibodies (against human CD2/CD3/CD28) using the T cell activation/expansion kit from Miltenyi Biotech GmbH (CA, USA). anti-biotin MACSiBead Particles loaded with the biotinylated antibodies are used to mimic antigen-presenting cells and activate T cells from PBMCs. Briefly, loaded anti-biotin MACSiBead™ were added to PBMCs at a T-cell/bead ratio of 1:2, and incubated for 2 days at 37 °C, and 8% CO2, according to the manufacturer’s instructions (Miltenyi Biotech GmbH, CA, USA). After removal of the beads by a magnetic device, these T cells were washed and added to the dermis compartment of lesional reconstructed skin models.
5
Cytokine-Induced Activation of Human Colonic Immune Cells
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Human colonic LPMCs were exposed to 50 ng/mL IL-23 or IL-1β (R & D Systems), or the combination of both, or IL-2 or IL-7 (Tonbo) for 16 h at 37°C + 5% CO2 followed by the addition of Golgi Plug Transport Inhibitor (BD Bioscience) for 4 h. For NKp44 activation experiments; 20 ug/mL of anti-NKp44-biotin (clone P44-8, Biolegend) was combined with Anti-Biotin MACSiBead Particles (Miltenyi Biotec) according to manufacturer's instructions. The beads were then added to LPMCs at a ratio of 5 beads to 1 LPMC for 16 h at 37°C + 5% CO2 followed by the addition of Golgi Plug Transport Inhibitor (BD Bioscience) for 4 h. Cells were then collected for flow cytometry as described below.
6
Activation and Cytokine Secretion Assay
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Anti-Biotin MACSiBead Particles (Miltenyi, Germany) were loaded with different activating antibodies. 1 × 105 cells and 0.5 × 105 antibody-loaded beads were added to a 96-well flat bottom plate (Cellstar, Greiner-Bio One, Austria) in a complete medium containing anti-CD107a (Supplementary Table 2). After 1 hour of incubation, monensin (Invitrogen, USA) was added to all wells, and incubation continued for 4 additional hours. Cells were harvested and washed, followed by intracellular staining with anti-IFNγ (Supplementary Table 2) using the Cytofix/Cytoperm Fixation/Permeabilization Kit (BD, USA) according to manufacturer’s protocol. Measurements were performed on a Navios EX flow cytometer (Beckman Coulter, USA). Data were analyzed using FlowJo software (v10.7.1, BD, USA).
7
Efficient Ex Vivo Expansion of Tumor-Infiltrating Lymphocytes
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TIL cultures were stimulated for 7 days with anti-biotin MACSiBead Particles loaded with biotinylated CD3/CD28 antibodies (Miltenyi Biotec, Bergisch Gladbach, Germany). The preparation of the conjugated beads was carried out according to the manufacturer’s recommendation (activation/expansion kit, Miltenyi Biotec). Antibody-loaded anti-biotin MACSiBead™ were added to the TIL at a cell/bead ratio of 1:1. For plate-bound expansion, 24-well plates were coated with 0.3 ml PBS per well containing 2 μg/ml anti-CD3 (clone OKT-3; Miltenyi Biotech), 2 μg/ml anti-CD28 (clone: 15E8, Miltenyi Biotech) with or without addition of 5 µg/ml CCL21+ 50 µg/ml ICAM1 and incubated overnight at 4°C. The following day, plates were washed with PBS, 0.01 x106 TIL in 2 ml CM medium with 3,000 IU/ml IL-2 were added per well for stimulation.
8
Isolation and Co-culture of MDSCs and T Cells
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Both PMN-MDSCs (by positive selection of Ly6G) and M-MDSCs (by positive selection of Gr1 of Ly6G depleted cell suspension) were isolated from the obtained single cell suspensions of the lung and spleen using the magnetic-activated cell sorting (MACS) MDSC isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany) according to manufacturer’s instructions. CD4+ and CD8+ T cells were isolated from the spleens of naïve BALB/c mice using MACS T cell isolation kits (Miltenyi Biotec) by negative selection according to manufacturer’s protocol. Isolated T cells were then stained with 1 µmol/L carboxyfluorescein succinimidyl ester (CFSE; BioLegend). CFSE-labeled CD4+ and CD8+ T cells were seeded in U-shaped 96-well plates at 1x105 cells per well and co-cultured with isolated PMN- or M-MDSCs at different ratios. Culture media consisted of RPMI 1640 supplemented with 10% FBS, 2 mM L-glutamine, 1% penicillin/streptomycin, 50 µM 2-mercaptoethanol together with 100 U/ml IL-2 (BioLegend) and anti-biotin MACSiBead particles loaded with CD3ϵ- and CD28-biotin (Miltenyi Biotec) with a bead-to-cell ratio of 1. After three days of co-culture the proliferation of CFSE-labeled CD4+ and CD8+ T cells was analyzed by flow cytometry (Supplementary Figure 3
9
T Cell Proliferation Assay with MSCs
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Proliferation of activated T cells was assessed as a measure of T cell deactivation. Initially, 5 × 104 MSCs were cultured in 96-well plates for 24 hours at 37 °C. CD4+ T cells were purified from spleens and lymph nodes (popliteal/inguinal) of healthy C57Bl/6 mice using the CD4+ T Cell Isolation Kit (Miltenyi) following manufacturer’s instructions. T cells were seeded at a density of 5.0 × 105/well in 250 μl RPMI medium with 10% FBS and MSCs (ratio 10:1) or with CM-MSC for 5 days. T cells alone served as control. Cells were activated using anti-Biotin MACSiBead Particles (Miltenyi) (ratio 2:1). Proliferation was assessed through reduction in signal intensity using VPD450 Violet proliferation dye (BD Biosciences) following previously described methodologies to calculate proliferative index and proliferative cycles24 (link),25 (link).
10
Th Cell Suppression by Regulatory T Cells
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Th cells and Tregs were sorted from PBMCs using a human CD4+CD25+Regulatory T Cell Isolation Kit (MACS, Miltenyi Biotec). Th cells were labeled with carboxyfluorescein succinimidyl ester (CFSE, Molecular Probes) according to manufacturer’s protocol. CFSE-labeled Th cells were stimulated with Anti-Biotin MACSiBead™ Particles (MACS, Miltenyi Biotec) and co-cultured with unlabeled Tregs. Th cell division was quantified based on serial halving of CFSE intensity, using algorithms provided by FlowJo software (Treestar).
Cytokine measurements in supernatants of co-cultures were performed using the Bio-Plex Pro Human Cytokine 17-plex Panel (Biorad), run on a Luminex 100 System (Luminex Corporation), according to manufacturer’s protocol.
Cytokine measurements in supernatants of co-cultures were performed using the Bio-Plex Pro Human Cytokine 17-plex Panel (Biorad), run on a Luminex 100 System (Luminex Corporation), according to manufacturer’s protocol.
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